The present invention relates to the measurement of the progress of a chemical reaction that generates an electroactive reaction product that is subsequently detected at an electrode amperometrically or coulometrically. The method is useful in applications where it is desirable to follow the progress of a chemical reaction, particularly in sensor applications where the progress of the reaction of an analyte can be useful in determining the analyte concentration.
In amperometric electrochemistry the current flowing at the electrode can be used as a measure of the concentration of electroactive species being reacted electrochemically at the working electrode. In coulometry the current flowing at the electrode is integrated over time to give a total amount of charge passed which yields a measure of the amount of electroactive material reacted at the working electrode. The current flowing (or charge passed at any time) at the electrode is dependent upon the rate of transfer of the electroactive species to the working electrode. When a significant concentration of electroactive species is situated close to the electrode and an electrical potential is applied to the electrode sufficient to electrochemically react the electroactive species at the electrode/solution interface, initially a higher current will flow which will diminish with time. For an isolated electrode, where the potential applied to the electrode is sufficient to react the electroactive species effectively instantaneously upon arriving at the electrode and the transfer of electroactive species to the electrode is controlled by diffusion, the current will follow a curve known in the art as the Cottrell Equation. According to this equation the current varies inversely with the square root of time. This yields a current which decays with time as the electroactive species that reacts at the electrode becomes depleted close to the electrode and so electroactive species has to travel from further and further away to reach the electrode as time progresses.
If in addition to the electrochemical reaction of the electroactive species at the electrode the electroactive species is being generated close to the working electrode by a chemical reaction, the form of the current flowing at the electrode becomes complex. The electrode reaction tends to decrease the concentration of electroactive species close to the working electrode whereas the chemical reaction tends to increase the concentration of the electroactive species in this region. The time dependent behavior of these two processes therefore mix and it is difficult to measure the chemical reaction kinetics from the current flowing (or charge passed) at the electrode.
For this reason, in the published literature, the rates of chemical reactions are not generally measured electrochemically except in specialized applications using specialized equipment. An example of such equipment is known in the art as a rotating ring/disc electrode. This apparatus is only applicable to relatively fast reaction kinetics and requires that the electrode be rotated at a known controlled rate with well characterized liquid hydrodynamics.
The method of the present invention allows for the extraction of chemical reaction rate information using a simple electrochemical method and apparatus.
In a first aspect of the present invention, a method is provided for measuring a rate of a chemical reaction between a component of a liquid sample and a reagent, the reaction producing an electroactive species, including providing an electrochemical cell having a working electrode, a counter electrode, and at least one wall; substantially immobilizing the reagent in the electrochemical cell at a site at a minimum distance from the working electrode, wherein the distance is such that transfer of the electroactive species from the site to the working electrode is diffusion controlled; placing the liquid sample in the electrochemical cell such that the liquid sample is in contact with the reagent, the working electrode, and the counter electrode; reacting the component with the reagent to produce the electroactive species; applying a potential between the working electrode and the counter electrode, wherein the potential is sufficient to electrochemically react the electroactive species at the working electrode; and measuring the current produced by the electrochemical reaction at the working electrode to obtain a measure of the rate of the chemical reaction.
In one aspect of this embodiment, the working electrode and the counter electrode are sufficiently spaced such that a product of an electrochemical reaction occurring at the counter electrode does not reach the working electrode while the current is measured. The working electrode and the counter electrode may be spaced apart at a distance greater than about 500 microns; between about 500 microns and about 5 mm; or between about 1 mm and about 2 mm. The working electrode and the counter electrode may be situated on the same plane.
In another aspect of this embodiment, the site and the working electrode are separated by a minimum distance ranging from about 10 microns to about 5 millimeters; from about 50 microns to about 500 microns; or from about 100 microns to about 200 microns.
In another aspect of this embodiment, the counter electrode is capable of functioning as a combined counter/reference electrode. The electrochemical cell may further include a reference electrode.
In another aspect of this embodiment, the working electrode functions as an anode, and may include platinum, palladium, carbon, carbon in combination with one or more inert binders, iridium, indium oxide, tin oxide, indium in combination with tin oxide, and mixtures thereof.
In another aspect of this embodiment, the working electrode functions as an cathode and may include platinum, palladium, carbon, carbon in combination with one or more inert binders, iridium, indium oxide, tin oxide, indium in combination with tin oxide, steel, stainless steel, copper, nickel, silver, chromium, and mixtures thereof.
In another aspect of this embodiment, the counter electrode includes platinum, palladium, carbon, carbon in combination with inert binders, iridium, indium oxide, tin oxide, indium in combination with tin oxide, steel, stainless steel, copper, nickel, chromium, silver, and mixtures thereof. The counter electrode may also include silver coated with a substantially insoluble silver salt, such as silver chloride, silver bromide, silver iodide, silver ferrocyanide, and silver ferricyanide.
In another aspect of this embodiment, the site is situated on the counter electrode or on the wall. The site and the working electrode may be situated on the same plane or in a plane facing and substantially parallel to the working electrode.
In another aspect of this embodiment, the reagent is contained within a polymeric matrix attached to a surface in the electrochemical cell; is chemically tethered to a surface in the electrochemical cell; is physically tethered to a surface in the electrochemical cell; or is dried onto a surface in the electrochemical cell and exhibits sufficiently low mobility in the liquid sample such that the reagent does not substantially migrate while the current is measured.
In another aspect of this embodiment, the method further includes a redox mediator. The redox mediator may include ferrocinium, osmium complexes with bipyridine, benzophenone, and ferricyanide.
In another aspect of this embodiment, the sample may include whole blood. The component may include glucose. The reagent may include an enzyme such as PQQ dependent glucose dehydrogenase, NAD dependent glucose dehydrogenase, glucose oxidase, lactate dehydrogenase, and alcohol dehydrogenase.
In another aspect of this embodiment, the potential is preferably between +50 and +500 mV, and more preferably about +300 mV.
In a second embodiment of the present invention, a method is provided for measuring a rate of a chemical reaction between glucose and PQQ dependent glucose dehydrogenase in whole blood including providing an electrochemical cell having a working electrode, a counter electrode, at least one wall, a redox mediator including ferricyanide and contained within the electrochemical cell, and a reagent including PQQ dependent glucose dehydrogenase, the reagent being substantially immobilized in the electrochemical cell at a site at a minimum distance from the working electrode; placing the whole blood sample in the electrochemical cell such that the sample is in contact with the reagent, the redox mediator, the working electrode, and the counter electrode; reacting the glucose with the PQQ dependent glucose dehydrogenase to produce reduced PQQ dependent glucose dehydrogenase, the reduced PQQ dependent glucose dehydrogenase in turn reacting with the ferricyanide redox mediator to form ferrocyanide; applying a potential between the working electrode and the counter electrode, wherein the potential is sufficient to electrochemically react the ferrocyanide at the working electrode; and measuring the current produced by the electrochemical reaction of ferrocyanide at the working electrode, wherein the measurement is indicative of the rate of the chemical reaction between glucose and PQQ dependent glucose dehydrogenase.